BIODIVERSITAS ISSN: 1412-033X Volume 22, Number 6, June 2021 E-ISSN: 2085-4722 Pages: 3326-3332 DOI: 10.13057/biodiv/d220638 Isolation and identification of thermophilic amylolytic bacteria from Likupang Marine Hydrothermal, North Sulawesi, Indonesia ELVY LIKE GINTING1,♥, LETHA L. WANTANIA2, EMMA MAUREN MOKO3, REINY A. TUMBOL1, MAYSE S. SIBY1, STENLY WULLUR1 1Faculty of Fisheries and Marine Science, Universitas Sam Ratulangi. Jl. Kampus Unsrat, Manado 95115, North Sulawesi, Indonesia. Tel./fax.: +62-431-868027, email: [email protected] 2Faculty of Mathematics and Natural Sciences, Bonn University. D-53012 Bonn, Germany 3Department of Biology, Faculty of Mathematics and Natural Science, Universitas Negeri Manado. Jl. Raya Tondano, Minahasa 95618, North Sulawesi, Indonesia Manuscript received: 1 April 2021. Revision accepted: 24 May 2021. Abstract. Ginting EL, Wantania LL, Moko EM, Tumbol RA, Siby MS, Wulur S. 2021. Isolation and identification of thermophilic amylolytic bacteria from Likupang Marine Hydrothermal, North Sulawesi, Indonesia. Biodiversitas 22: 3326-3332. The aims of the research were to isolate and identify the amylase-producing thermophilic bacteria from Likupang Marine Hydrothermal, North Sulawesi, Indonesia. The bacteria were characterized based on the colony and cell morphology and subsequently screened for their amylase activities. The bacterial isolates were identified based on 16S rRNA gene sequences. There were 12 thermophilic bacteria isolates from Likupang Marine Hydrothermal that were able to produce amylase. Two selected isolates (L3 and L9) had an amylolytic index value in the range of 3.04-3.52 at 55oC. The colonies of L3 and L9 are circular, and they are Gram positive, rod-shaped, and motile bacteria. Based on the 16S rRNA gene sequences and phylogenetic tree analysis showed that L3 was closely related to Bacillus caldotenax with 93% similarity while L9 had 99% similarity to B. caldotenax. Hence, we identified L3 isolate as Bacillus sp. strain L3 and L9 isolate as B. caldotenax strain L9. Both of these bacteria were grouped as Geobacillus thermoleovorans. Keywords: Amylase, Bacillus, identification, Likupang, thermophilic Abbreviations: BLAST: Basic local alignment search tool, DDH: DNA-DNA hybridization, DNA: deoxyribonucleic acid, PCR: polymerase chain reaction, TMMB: thermus medium modified broth, TMMA: thermos medium modified agar, 16S rRNA: 16 Svedberg ribosome-ribonucleic acid, VP: Voges-Proskauer INTRODUCTION thermostability of cell components in high and elevated temperatures is the reason for their extensive use in Bacterial populations are widespread in various industrial applications and multiple biotechnological environmental conditions, including many places with (Raddadi 2015; Charbonneau et al. 2012). extreme conditions, such as pH, salinity, and temperature. In high-temperature bioprocessing, the enzymes Bacteria that can survive in high temperatures are known as produced by thermophilic bacteria can reduce risk of thermophilic bacteria. One of the notable abilities of contamination, improve solubility of substrates (i.e., thermophilic bacteria is to produce enzymes that are stable lignocellulosic biomass), lower chances of phage infection, at high temperatures, and the enzymes catalyze their and reduce cooling costs. The cooling cost specifically are chemical reactions at high speed at high temperatures reduced due to greater temperature differential between the (Singh et al. 2011; Mohammad et al. 2017). Moreover, fermenter and the ambient air, which is the ultimate heat thermophilic bacteria are the ideal choice for producing acceptor with the addition of continuous recovery of thermostable enzymes, which makes them interesting volatile chemical products directly from fermentation research subjects. In particular, the thermostability of their broth. (Frock and Kelly 2012; Keller et al. 2014). One of cell components in highly elevated temperatures has led to the enzymes produced by thermophilic bacteria is amylase their extensive use in various biotechnological and which has an important role in biotechnological studies and industrial applications (Raddadi 2015; Charbonneau et al. the world enzyme market (25%-33%) (Nguyen et al. 2002). 2012). They were very useful in industrial application Amylase hydrolyze starch to produce maltose, glucose, because they have several advantages, i.e., high growth malto-oligosaccharides, and various α-limit dextrin- rate, accelerate fermentation processes two to three times containing α (1-6) bonds. They have a spacious range of over those with mesophilic producers, reduced risk of uses in pharmaceutical, cosmetic, agricultural industries, microbial contamination, higher diffusion rate, and mass food, and nutritional processes (Satrimafitrah et al. 2019; turnover, reduced the viscosity of culture liquids, and Gazali and Suwastika 2018; Luang-In et al. 2019; De improved solubility of polymeric substrates and fats that Souza and Magalhaes 2010). are usually insoluble compounds (Kambourova 2018). The GINTING et al. – Identification of thermophilic amylolytic bacteria 3327 Some hot springs in Indonesia are sources of isolation and further testing. The temperatures during the thermophilic bacteria that could produce amylase (Gazali et sampling were measured on site using glass thermometer. al. 2018; and Ardhi et al. 2020). Thermophilic bacteria can also be found in shallow water hydrothermal and seabed Culture and bacteria isolation hot springs. Marine hydrothermal spots can be discovered Twenty µL of marine water sample was inserted into 5 in many places throughout Indonesia including North mL of Thermus Medium Modified Broth (TMMB-0.01% Sulawesi. One of the marine hydrothermal in North MgSO4.7H2O, 0.1% K2HPO4, 0.35% (NH4)2SO4, 0.1% Sulawesi is in Likupang. A study by Myung-Ji et al. (2012) NaCl, 0.05% Yeast extract, 0.05% peptone) and incubated revealed microbial diversity in Likupang based on the 16S at 55oC for 24-48 hours. As much as 0.1 mL of cultured rRNA gene sequence. One hundred and five representative broth was spread onto TMMA (TMMB + 2% Agar) using clones from this thermal vent showed that the highest L-glass followed by incubation at 55oC for 24-48 hours. proportion was γ-Proteobacteria (33.3%), followed by Single growing colonies with different morphological Bacteroidetes (27.6%) and unclassified bacteria group characteristics (e.g., color, colony, growth shape, and (20%). The minor groups were affiliated to Firmicutes edges) were individually isolated, which were then (7.6%) and α-Proteobacteria (4.8%). On the other hand, the developed on new TMMA, and observed more carefully research on amylase-producing thermophilic bacteria from for their morphology characteristics. this area has not been reported yet. Therefore, the present study was carried out to isolate and identify the Screening the thermopiles bacteria producing amylase thermophilic amylolytic bacteria from Likupang Marine The single colony of the bacterium was spotted onto the Hydrothermal, North Sulawesi, Indonesia. surface of TMMA containing 2% of amylum on a plate, followed by incubation at 55oC for 24-48 hours. The ability of the bacteria to produce amylase was observed by the MATERIALS AND METHODS appearance of a clear zone around the colony by flooding it with Lugol’s iodine solution (w/v) (1% iodine in 2% Sampling potassium iodide). The clear zone and colony diameters Marine water samples were collected from 3 points on were estimated using a caliper rule (Poletto et al. 2018). the hot spring location at East Likupang beach, North The amylolytic index value of each isolate was determined Minahasa District, North Sulawesi Province, Indonesia based on the ratio of clear zone diameter and diameter of a (1o39'54.87''N, 125o06'19.41''E) (Figure 1). They were colony (Soy et al. 2019). taken directly using sterile bottles and placed in cool box. Samples were carried to the laboratory for bacteria Figure 1. The map of North Minahasa District, North Sulawesi Province, Indonesia. The sampling location is in the Likupang Marine Hydrothermal, East Likupang Sub-district as indicated by an arrow 3328 BIODIVERSITAS 22 (6): 3326-3332, June 2021 Identification of the active amylolytic thermophilic bacteria amylolytic index at TMMA containing 2% of amylum. Morphological and physio-biochemical characterization There were two isolates (L3 and L9) that have the highest Preliminary identification of the bacterial isolates with amylolytic indexes were further identified. amylolytic activity was carried out based on morphological L3 and L9 had different morphological and physio- and physio-biochemical characteristics. Morphological biochemical characteristics (Table 1). The ability of these characteristics observed were Gram-stain (Thairu et al. bacteria to degrade amylum is indicated by the appearance 2014), motility of bacteria, and biochemical properties. of a clear zone around the colonies on the media containing Bacterial motility was determined using semi-solid media, 2% amylum at 55oC (Figure 2). The average amylolytic while biochemical properties included parameters of index of L3 and L9 were 3.04 and 3.52, respectively. The carbohydrate utilization, gas production, sugar more amylase, the wider the clear zone due to the fermentation (glucose, lactose, sucrose, maltose), indole degrading of amylum on the medium which renders production, methyl red, Voges-Proskauer (VP), citrate enhancement the amylolytic index. utilization, and H2S production. The 16S rRNA gene of the two